Chemical libraries, such as combinatorial chemical libraries, comprise chemical compounds that have been synthesized from a systematic series of reactions. Such libraries can include an extraordinarily large and varied collection of compounds. Large chemical libraries are typically used to screen for biological activity, such as for pharmaceutical and agricultural purposes, among others.
Chemical libraries are typically created by either "parallel synthesis" or "split synthesis." In the former method, different compounds are synthesized in separate vessels. A commonly used format for parallel synthesis is the 96-well microtiter plate. An automated process can be used for adding different reagents to separate wells of the plate in a predefined manner to produce a chemical library.
In the split synthesis method, compounds are assembled on the surfaces of polymeric resin beads or other solid supports by an iterative process. In particular, a sample of beads is divided among several reaction flasks, and a different compound or "chemical building block" is added to each flask. The chemical building blocks link to the beads, one to each bead. The beads from all the containers are pooled, divided into a new set of reaction flasks, and a different chemical compound is added to each container. The added compound reacts with the building blocks already linked to each bead. The beads are again pooled, divided and a new group of compounds are added, one to a container. Reaction occurs, and the process is repeated as desired. A very large and diverse chemical library may be obtained after relatively few such iterations.
Comparing the methods, split synthesis typically yields relatively smaller quantities of a relatively larger number of compounds, whereas parallel synthesis generates relatively larger quantities of a relatively smaller number of compounds. Constructing a library by either method may involve determining and optimizing reaction conditions to yield desired products, analyzing reaction products to assure that desired products are obtained, as well as determining the yield and purity of such products.
Whether optimizing a large split-synthesis-derived chemical library, or simply analyzing the products of a large parallel synthesis-derived chemical library, the time and resources expended for qualitative and quantitative analysis thereof is substantial. While conventional chemical analysis techniques and devices can be used for analyzing chemical libraries, such methods are, in practice, unworkable, when the library contains a large number of compounds.
It is known in the art that an evaporative light scattering detector (ELSD) may be advantageously used in conjunction with high performance liquid chromatography (HPLC) to quantitate compounds associated with chemical libraries. ELSD is particularly well suited to the quantitation (e.g., yield determination, etc.) of individual compounds of chemical libraries because it is a "universal" detector, i.e., its response is substantially independent of specific physical or chemical properties of the compound being analyzed. As such, the ELSD response may be calibrated by a single standard, rather than a large number of reference standards corresponding to each compound being analyzed, which, for large chemical libraries, would be unworkable.
Note that unlike the ubiquitous UV absorbance detector, the response of the ELSD is not dependent on the presence of a chromophore in the analyzed compound. Chromophores vary from compound to compound, and the UV absorbance detector responds differently to different chromophores. As such, a large number of standards are disadvantageously required when using a UV absorbance detector for analyzing a large number and variety of compounds. Moreover, chromophores may be absent in many of the compounds present in chemical libraries, frustrating the detection of such compounds via UV absorbance. For additional background concerning the shortcomings of common analytical techniques as applied to the identification, purification and quantification of chemical libraries, see U.S. Pat. No. 5,670,054, incorporated by reference herein.
An example of using ELSD in a technique for identifying, purifying and quantitating library-derived chemical compounds is provided by Kibbey et al. in the aforementioned U.S. Pat. No. 5,670,054. According to the patent, a first HPLC column is used in conjunction with a mass spectrograph to characterize compounds present in a mixture. The chromatographic and mass spectroscopic data generated by the mass spectrograph are used to purify several compounds of interest from the mixture through a system including a second HPLC column and a UV absorbance detector. Finally, the concentration of each compound of interest, as purified, is estimated using a system that includes an HPLC column and an ELSD.
While advantageously using an ELSD for quantitation, the Kibbey et al. method for purifying chemical-library-derived compounds requires multiple runs through different HPLC columns connected to different analytical devices. While such an approach does appear to provide a way to purify library-derived compounds, it does not provide the art with a high-throughput method for rapid analysis of a large number of compounds, such as is useful, for example, for optimizing and/or analyzing chemical libraries. Until now, such a method and system has been unavailable.